Bottom Line:
Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99.To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies.The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

ABSTRACTTwo outstanding questions concerning antinuclear antibodies (ANAs) in lupus involve their pathogenic potential and their molecular signatures. To address these questions, a panel of 56 antinuclear and 47 nonnuclear binding monoclonal antibodies was rescued from four seropositive NZM2410 lupus mice. The monoclonals varied in their reactivity to nucleosomes, ssDNA, dsDNA, and glomerular substrate. A large fraction of the antibodies demonstrated apparent polyreactivity (to DNA, histones, and glomerular antigens) due to bound, DNase-1 sensitive nuclear antigenic bridges. Although nephrophilic immunoglobulin (Ig) M and IgG antibodies were the most pathogenic, the dsDNA-binding antibodies were modestly so; in contrast, antinucleosome antibodies were clearly not pathogenic. Compared with the nonnuclear antigen-binding monoclonal antibodies rescued from the same mice, ANAs exhibited increased utilization of VH5/7183 genes and highly cationic heavy chain (HC) CDR3 regions. Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99. To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies. The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

fig4: Renal pathogenicity of NZM2410-derived IgM ANAs. (A) Nine representative NZM2410-derived IgM mAbs were tested for in vivo pathogenicity as detailed in Materials and Methods. The specificity profiles of these Abs that have been tabulated below the figure were obtained from Table I. It should be noted that “+” in this figure simply indicates that the mAb does react with the respective Ag, independent of the strength of reactivity. Both the beginning (D0, white dots) and ending (D10, black dots) 24-h urine protein levels (measured using metabolic cages) are depicted. Where the D10 proteinuria levels were found to be significantly higher than the corresponding D0 levels (using the Student's t test), the p-values are listed. (B) Depicted below are the BUN levels measured on D0 and D10 after Ab administration. Depicted below each column of dots are the p-values (calculated using the Student's t test), where the D0 and D10 BUN values were compared and found to be significantly different.

Mentions:
Next, a panel of nine IgM mAbs was selected for pathogenicity testing. As aforementioned, uninjected NZW mice excreted urinary protein of ≥1 mg/d. Clearly, IgM non-ANAs and Abs with reactivity restricted to histone–DNA nucleosomal complexes and/or ssDNA (but not dsDNA) did not compromise renal function significantly (Fig. 4 A). In contrast with the findings with the IgG ANAs, IgM Abs that were discordant in their reactivity patterns to dsDNA and glomeruli (e.g., ZA7F8, ZDC3, and ZA5E11) did not appear to be pathogenic. However, IgM Abs that had the potential to bind all of the aforementioned Ags, including dsDNA and glomerular substrate, were clearly pathogenic, leading to the excretion of 1–1.6 mg/d urinary protein (Fig. 4 A). Indeed, the D10 24-h urinary protein levels in the mice injected with these apparently polyreactive IgM Abs (n = 13; mean = 1.3 mg/24-h) were significantly higher than the D10 24-h urinary protein excretion levels observed in all other experimental groups combined (n = 30; mean = 0.9 mg/24-h; P < 0.0006). Finally, the mice in two of the three groups receiving dsDNA/glomeruli dual-reactive IgM Abs exhibited significantly elevated BUN levels at the conclusion of the experiments (Fig. 4 B).

fig4: Renal pathogenicity of NZM2410-derived IgM ANAs. (A) Nine representative NZM2410-derived IgM mAbs were tested for in vivo pathogenicity as detailed in Materials and Methods. The specificity profiles of these Abs that have been tabulated below the figure were obtained from Table I. It should be noted that “+” in this figure simply indicates that the mAb does react with the respective Ag, independent of the strength of reactivity. Both the beginning (D0, white dots) and ending (D10, black dots) 24-h urine protein levels (measured using metabolic cages) are depicted. Where the D10 proteinuria levels were found to be significantly higher than the corresponding D0 levels (using the Student's t test), the p-values are listed. (B) Depicted below are the BUN levels measured on D0 and D10 after Ab administration. Depicted below each column of dots are the p-values (calculated using the Student's t test), where the D0 and D10 BUN values were compared and found to be significantly different.

Mentions:
Next, a panel of nine IgM mAbs was selected for pathogenicity testing. As aforementioned, uninjected NZW mice excreted urinary protein of ≥1 mg/d. Clearly, IgM non-ANAs and Abs with reactivity restricted to histone–DNA nucleosomal complexes and/or ssDNA (but not dsDNA) did not compromise renal function significantly (Fig. 4 A). In contrast with the findings with the IgG ANAs, IgM Abs that were discordant in their reactivity patterns to dsDNA and glomeruli (e.g., ZA7F8, ZDC3, and ZA5E11) did not appear to be pathogenic. However, IgM Abs that had the potential to bind all of the aforementioned Ags, including dsDNA and glomerular substrate, were clearly pathogenic, leading to the excretion of 1–1.6 mg/d urinary protein (Fig. 4 A). Indeed, the D10 24-h urinary protein levels in the mice injected with these apparently polyreactive IgM Abs (n = 13; mean = 1.3 mg/24-h) were significantly higher than the D10 24-h urinary protein excretion levels observed in all other experimental groups combined (n = 30; mean = 0.9 mg/24-h; P < 0.0006). Finally, the mice in two of the three groups receiving dsDNA/glomeruli dual-reactive IgM Abs exhibited significantly elevated BUN levels at the conclusion of the experiments (Fig. 4 B).

Bottom Line:
Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99.To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies.The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

ABSTRACTTwo outstanding questions concerning antinuclear antibodies (ANAs) in lupus involve their pathogenic potential and their molecular signatures. To address these questions, a panel of 56 antinuclear and 47 nonnuclear binding monoclonal antibodies was rescued from four seropositive NZM2410 lupus mice. The monoclonals varied in their reactivity to nucleosomes, ssDNA, dsDNA, and glomerular substrate. A large fraction of the antibodies demonstrated apparent polyreactivity (to DNA, histones, and glomerular antigens) due to bound, DNase-1 sensitive nuclear antigenic bridges. Although nephrophilic immunoglobulin (Ig) M and IgG antibodies were the most pathogenic, the dsDNA-binding antibodies were modestly so; in contrast, antinucleosome antibodies were clearly not pathogenic. Compared with the nonnuclear antigen-binding monoclonal antibodies rescued from the same mice, ANAs exhibited increased utilization of VH5/7183 genes and highly cationic heavy chain (HC) CDR3 regions. Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99. To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies. The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.